The use of transgenic mosquitoes in an integrated malaria control strategy will require mosquitoes that completely block parasite development, yet remain competitive with wild mosquito populations. Manipulation of key signaling cascades regulating both immunity and fitness represents a novel approach to achieving this goal. In mosquitoes and other model invertebrates, the midgut functions as a center for insulin/insulin growth factor signaling (IIS), which controls immunity, lifespan, metabolism, and reproduction. The effects of midgut IIS are largely mediated through mitochondrial dynamics and activity, defined as mitochondrial biogenesis, bioenergetics, and clearance of damaged mitochondria through mitophagy. In invertebrates and mammals, IIS- dependent mitochondrial dynamics and mitochondrial metabolism regulate a wide range of important physiologies, including epithelial barrier integrity, stem cell maintenance and differentiation, lifespan and immunity, indicating tha this regulation is fundamental in living organisms. Through our work with Anopheles stephensi, we discovered that manipulation of IIS in the midgut alters the critical balance of mitochondrial dynamics and activity, resulting in phenotypic changes in mosquito resistance to Plasmodium falciparum infection as well as to mosquito lifespan and reproduction. Thus, we propose that IIS-dependent mitochondrial dynamics and activity control midgut health in A. stephensi, which underlies the effects of IIS on immunity, lifespan, metabolism, and reproduction. To define how IIS-dependent mitochondrial function regulates these important phenotypes, we will use five distinct treatments - Akt overexpression (increased IIS), PTEN overexpression (decreased IIS), provisioning with human insulin or IGF1, and manipulation of endogenous A. stephensi insulin-like peptides (AsILPs) - to push and pull mitochondrial dynamics and activity in the midgut. Specifically, we will define how midgut IIS-dependent mitochondrial biogenesis, bioenergetics, oxidative phosphorylation, and mitophagy regulate stem cell maintenance and differentiation, epithelial integrity, and cell death processes to control fitness and Plasmodium resistance. From these studies, we will identify and manipulate specific gene targets that directly regulate mitochondrial function to retain parasite resistance while concurrently enhancing mosquito fitness. We will overexpress four of these candidate genes based on our observations and published studies and two candidate genes identified from Aims 1 and 2 in the midgut singly or in pairs. Our goal for this project is to generate highly fit, P. falciparum resistant A. stephensi that can be deployed for malaria control. In the longer term, this transgenic platform could also be additive with other gene drivers and customized with anti-parasite effectors for sustainable resistance management.